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Mechanism of actin filament nucleation by the bacterial effector VopL

Nature Structural & Molecular Biology volume 18, pages 1068–1074 (2011)Cite this article

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Abstract

Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.

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Figure 1: Both the WH2 motifs and VCD contribute to actin nucleation by VopL.
Figure 2: Dimerization is necessary for actin nucleation by the VopL WH2 motifs.
Figure 3: Structure of the VCD dimer.
Figure 4: The VCD contributes to actin assembly activity by dimerization and contacts to actin.
Figure 5: Increasing the linker between WH2c and the VCD increases actin assembly activity.
Figure 6: Model for a minimal actin nucleus assembled by VopL.

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Acknowledgements

We thank J. Zahm of the University of Texas Southwestern Medical Center (UT Southwestern) for the data in Supplementary Figure 7b and for discussion; Z. Otwinowski, D. Borek (UT Southwestern) and participants in the 2010 CCP4 workshop, especially P. Afonine (Lawrence Berkeley National Laboratory), G. Murshudov (The University of York) and B. Lohkamp (Karolinska Institute), for technical assistance with protein structure determination; F. Correa (UT Southwestern) for assistance with MALLS experiments and data analysis; and K. Orth, Z. Chen and S. Padrick for discussion. Results shown in this report are derived from work conducted at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. Work was supported by funds from the Howard Hughes Medical Institute and a Welch Foundation grant to M.K.R. (I-1544).

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Authors and Affiliations

  1. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Bingke Yu, Hui-Chun Cheng, Chad A Brautigam, Diana R Tomchick & Michael K Rosen

  2. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Bingke Yu, Hui-Chun Cheng & Michael K Rosen

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  1. Bingke Yu
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Contributions

M.K.R. conceived the project; B.Y. did the work; C.A.B. and D.R.T. assisted with X-ray diffraction data collection and structure determination. H.-C.C. assisted with designing and making the VopL constructs; M.K.R. and B.Y. analyzed the data and wrote the paper.

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Correspondence to Michael K Rosen.

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Yu, B., Cheng, HC., Brautigam, C. et al. Mechanism of actin filament nucleation by the bacterial effector VopL. Nat Struct Mol Biol 18, 1068–1074 (2011). https://doi.org/10.1038/nsmb.2110

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